1. Field of the Invention
The invention relates to a method and apparatus for analyzing isoenzymes. Isoenzymes are multiple molecular forms of an enzyme which catalyze the same reaction, but differ in certain physical chemical properties, such as electrophoretic mobility. Following electrophoresis five isoenzymes of lactic dehydrogenase (LDH) and three isoenzymes of creatine phosphokinase (CPK) have been demonstrated in human serum. Each isoenzyme of LDH is designated by a number related to its electrophoretic mobility. The fastest moving fraction (most anodic) is designated LDH1 and is found primarily in heart muscle. The slowest moving (most cathodic) is LDH5 and is found primarily in liver and skeletal muscle. The others LDH2, LDH3 and LDH4 are found to varying degrees along with LDH1 and LDH5 in all tissues.
The multiple forms of CPK demonstrated after electrophoresis are due to the fact that CPK is a dimer (two polypeptide chains). Both polypeptide chains of skeletal muscle CPK are the same and have been designated MM. Brain CPK, identified as BB, contains two identical polypeptide chains, but they differ from the polypeptide chains of MM. Heart muscle CPK contains one skeletal muscle and one brain polypeptide chain and is designated MB. During electrophoresis, BB moves fastest and is found closest to the anode. MM moves slowest and is found closest to the cathode. MB moves in between. The most important current clinical application for isoenzyme analysis is to confirm or rule out the diagnosis of acute myocardial infarction. A patient admitted to the hospital with chest pains should have CPK and LDH isoenzyme analyses run immediately upon admission. Another assay should be performed within 6-13 hours and 24-37 hours. If an increased CPK MB level cannot be demonstrated during this period, there is about a 100% certainty that the patient didn't sustain a myocardial infarct for the episode in question.
Normally, LDH2 is greater than LDH1. Following myocardial infarct, LDH1 increases, resulting in LDH1 becoming greater than LDH2. This is referred to as a "flipped LDH ratio". This occurs in 80% of all myocardial infarcts in 48 hours. The presence of an increased CPK MB and a flipped LDH ratio within the first 48 hours is almost 100% indicative of a myocardial infarction. Increased CPK MB and normal LDH may indicate intermediate syndrome, coronary insufficiency, or crescendo angina as well as myocardial infarct. Other measures, such as EKG must then be considered to establish the diagnosis.
2. Description of the Prior Art
Enzyme analyses are generally performed in solution by spectrophotometric or fluorometric measurements under controlled conditions of the disappearance or appearance of a substrate or its metabolite. Isoenzymes can be separated by column chromatography and the enzyme concentration of the eluates determined by such means. However these procedures have been shown to have poor precision and accuracy and to be fraught with problems of interpretation. They are also time consuming. In current clinical practice, isoenzymes are generally analyzed by first performing electrophoretic separation of the serum on a cellulose acetate or agarose support strip in an electric field in a suitable buffer for 10 minutes. The strip containing the separated isoenzymes is then sandwiched against a second strip holding the appropriate reagents to visualize either CPK or LDH after 25 minutes incubation. The strip is then dried and quantitated by a scanning fluorescent densitometer. 13 (CPK) or 20 (LDH) individual skilled steps are necessary before the strip is ready for the densitometer. The automatic densitometer costs $3750 to $6000 and a computer to assist its somewhat complex operation costs at least $3000. A considerable effort and time of a skilled operator is required for these densitometric measurements.
There may be other interfering enzymes or agents in the serum which may react to give false readings of the desired enzyme. It is expected that other isoenzyme analyses may prove useful in the future when suitable methods and data become available.
It is difficult to provide at all hours the skilled staff required to perform these analyses. With all the other duties charged to the laboratorian, such time consuming procedures cannot be performed on the stat basis that many patients' condition dictates. The present procedures are so awkward and involve so many steps and variables that they are quite imprecise even in the best of hands. A need exists for a technique which completes automation with simplicity, versatility, precision, and rapid throughput for stat samples that will be available for use by relatively unskilled persons at all hours with little operator time.